Polyvalent influenza virus-like particle (vlp) compositions

ABSTRACT

Polyvalent influenza virus-like particles (VLPs) comprising influenza antigenic polypeptides are described. Also described are compositions comprising these polyvalent VLPs as well as methods of making and using these VLPs.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 11/796,987, filed Apr. 30, 2007, which claims the benefit ofU.S. Provisional Application No. 60/796,738, filed May 1, 2006. Theentire disclosure of the above-referenced application is incorporatedherein by reference.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

Funds used to support some of the studies disclosed herein were providedby grant number 1 R43 AI063830-01 awarded by the National Institute ofAllergy and Infectious Disease (NIAID) of the National Institutes ofHealth (NIH). The U.S. Government may have certain rights in theinvention.

TECHNICAL FIELD

Multivalent virus-like particles (VLPs) containing influenza antigensare described, as are methods and making and using these VLPs.

BACKGROUND

The influenza A virus is a well characterized virus that infects humansas well as a large number of other species. See, e.g., U.S. PatentPublication No. 20050186621. All of the sixteen (Knossow M and Skehel J.J. (2006) Immunology 119(1):1-7) subtypes of influenza A virus circulatein wild birds and domestic avian species. Few influenza subtypes areepidemic among humans, but periodically pandemic strains derived fromanimals or birds unpredictably emerge causing wide spread disease ofhigh morbidity and mortality.

Currently, influenza vaccines are produced in fertilized chicken eggs.Eleven days after fertilization, a single strain of influenza virus isinjected into the eggs. The virus multiplies in the infected embryo andafter several days of incubation, the eggs are opened the virusharvested, purified, chemically inactivated for killed vaccine) andcombined with other similarly produced strains to generate an influenzavaccine. On average, one to two eggs are needed to produce one dose ofvaccine and the entire production process lasts at least six months.

Traditional influenza vaccines are trivalent in that they containantigenic proteins from three, different influenza virus strains (e.g.,two subtypes A and one subtype B). Accordingly, each antigenicallydistinct virus must be produced separately in embryonated eggs or tissueculture, isolated and then combined in the final vaccine formulationmeaning that one hundred million doses of vaccine necessitate the use ofone hundred million eggs to produce only one of the vaccine components.The production of the three virus components currently included in thevaccine requires the use of three hundred million eggs or has similarproduction requirements in a tissue culture production process. Giventhe long and costly production protocols, it is unlikely that egg-basedproduction of flu vaccines could be used to contain a flu pandemic.

Therefore, there remains a need for polyvalent influenza compositionsand methods that prevent and/or treat infection with the various highlyvirulent and transmissible influenza strains.

SUMMARY

Described herein are polyvalent virus-like particles (VLPs) comprisingtwo or more influenza proteins (e.g., antigens, structural proteins),preferably two or more antigenic influenza polypeptides from differentstrains. Also described are compositions comprising these VLPs, as wellas methods for making and using these VLPs. The polyvalent VLPspreferably comprise influenza matrix protein M1 and at least two HAantigens derived from different influenza virus strains and/or at leasttwo NA antigens derived from different influenza virus strains. Methodsof making and using these compositions are also described.

VLP are structures that morphologically resemble an influenza virus, butare devoid of the genetic material required for viral replication andinfection. Using VLPs rather than inactivated influenza virus for theproduction of polyvalent VLP vaccines has several advantages, includingease of production and purification, as compared current vaccines whichare manufactured in eggs. Influenza VLP vaccine compositions may alsoavoid or reduce the unwanted side effects of current inactivated,egg-based vaccines seen in young children, elderly, and people withallergies to components of eggs. Furthermore, unlike many inactivatedinfluenza virus vaccines, the HA and NA proteins of the VLPs describedherein maintain conformational epitopes involved in eliciting aprotective neutralizing antibody response. Using polyvalent VLPspresenting HA and/or NA proteins from multiple influenza strains allowsimmune responses to be generated to one or more influenza strains,reduces vaccine production costs, and allows vaccines to be safelyproduced to highly pathogenic viruses. Thus, the polyvalent VLPsdescribed herein provides for enhanced vaccine safety, coverage,efficacy and ease in manufacturing and allows for the safe creation ofpolyvalent vaccines against multiple human and avian influenza viruses.

In one aspect, provided herein is a polyvalent influenza VLP comprisingat least one influenza matrix protein (M1 and optionally M2) and two ormore antigenic influenza glycoproteins, wherein the antigenicglycoproteins are derived from two or more different influenza strains.In certain embodiments, the VLP includes a single matrix protein, forexample M1. In other embodiments, the VLP comprises M1 and M2. Incertain embodiments, the glycoproteins are selected from the groupconsisting of HA, NA and combinations thereof.

Any of the VLPs described herein may further comprise an influenzanucleoprotein (NP) and/or one or two proteins of the polymerase complex(made up of PB1, PB2 and PA). For example, the VLP may include NP and/orPB1, PB2 and PA; NP and/or PB1 and PB2; NP and/or PB1 and PA; and/or NPand/or PB2 and PA.

In any of the polyvalent VLPs described herein, the M1 protein maycomprise an amino acid modification as compared to a wild-type M1protein, for example, modifications to one or more of the following:modification of the nuclear localization signal (NLS); modification ofone or more determinants of spherical structure; modification of one ormore amino acid residues involved in protein-protein interactions;and/or introduction of one or more L-domains.

In any of the VLPs described herein, the M1 and/or optional M2 proteinmay comprise an amino acid modification as compared to a wild-typematrix protein, for example, introduction of one or more L-domainsand/or creation of a fusion of the matrix protein and animmunomodulatory polypeptide.

In yet another aspect, the disclosure provides a polyvalent VLP in whicha portion of one or more of the glycoproteins proteins of any of theVLPs described herein is replaced with a homologous region of aglycoprotein protein from a different influenza strain or subtype. Incertain embodiments, the transmembrane domain of the glycoprotein isreplaced. In other embodiments, the cytoplasmic tail region of theglycoprotein is replaced. In yet other embodiments, the transmembranedomain and the cytoplasmic tail region of one or more glycoproteins arereplaced with domains from one or more different influenza proteins.

In another aspect, described herein is a host cell comprising any of theVLPs as described above. The host cell may be an insect, plant,mammalian, bacterial or fungal cell.

In yet another aspect, a cell stably transfected with a sequenceencoding an influenza matrix protein or an influenza glycoprotein isprovided. The cell may be an insect, plant, mammalian, bacterial orfungal cell. In certain embodiments, the cell is a mammalian cell line.

In another aspect, a packaging cell line is provided for producinginfluenza VLPs as described herein. The cell line is stably transfectedwith one or more polynucleotides encoding less than all of the M1, HAand NA of the VLP (e.g., at least one of the influenza proteins formingthe VLP), and upon introduction and expression of the one or moreinfluenza protein-encoding sequences not stably transfected into thecell, the VLP is produced by the cell. In certain embodiments, sequencesencoding M1 and/or M2 are stably integrated into the packaging cell lineand sequences encoding the glycoproteins expressed on the surface of theVLP are introduced into the cell such that the VLP is formed. In otherembodiments, sequences encoding one or more of the glycoproteins arestably integrated into the cell to form a packaging cell line and VLPsare formed upon introduction of sequences encoding M1 and, optionally,M2. The packaging cell may be an insect, plant, mammalian, bacterial orfungal cell. In certain embodiments, the packaging cell is a mammaliancell line.

In yet another aspect, the disclosure provides an immunogeniccomposition comprising any of the VLPs described herein and apharmaceutically acceptable excipient. In certain embodiments, thecompositions further comprise one or more adjuvants.

In a still further aspect, a method of producing a VLP as describedherein is provided, the method comprising the steps of: expressing oneor more polynucleotides encoding the M1 and at least two influenzaglycoproteins in a suitable host cell under conditions such that theVLPs assemble in the host cell; and isolating the assembled VLPs fromthe host cell. The host cell can be a mammalian cell, an insect cell, ayeast cell or a fungal cell. In certain embodiments, an expressionvector comprising one or more polynucleotides operably linked to controlelements compatible with expression in the selected host cell areintroduced into the host cell. The expression vector may be a plasmid, aviral vector, a baculovirus vector or a non-viral vector. In certainembodiments, one or more of the polynucleotides are stably integratedinto the host cell. Alternatively, one or more of the polynucleotidesmay be transiently introduced into the host cell.

In another aspect, provided herein is a method of generating an immuneresponse in a subject to two or more influenza viruses, the methodcomprising the step of administering a composition comprising one ormore polyvalent VLPs as described herein to the subject. In certainembodiments, the composition is administered intranasally. Any of themethods may involve multiple administrations (e.g., a multiple doseschedule).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic depicting the structure of exemplary polyvalentsub-viral structures (VLPs) as described herein. The matrix M1 proteinunderlines the membrane of the structure and the M2 protein forms achannel across the sub-viral structural membrane. The surface of the VLPis decorated with multiple antigenically distinct glycoproteins.

FIG. 2 is a schematic depicting exemplary amino acid sequencemodifications to matrix protein 1 (M1). These modifications may enhanceassembly and/or release of polyvalent influenza VLPs by cellstransfected with constructs as described herein.

FIG. 3 is a schematic depicting exemplary amino acid sequencemodifications to matrix protein 2 (M2). These modifications may enhanceincorporation and/or potency of polyvalent influenza VLPs as describedherein.

FIG. 4 is a schematic diagram showing an exemplary polyvalent VLP asdescribed herein in which the transmembrane and/or cytoplasmic taildomains of the surface antigens (e.g., glycoproteins HA and/or NA) arereplaced with homologous domains from other influenza.

FIG. 5 is a schematic diagram of a baculovirus transfer vector carryingtwo antigenically distinct HA molecules.

FIG. 6, panels A and B, depict Western blot analysis of polyvalent VLPsprepared as described in Example 1. FIG. 6A shows that the VLPs compriseHA 1918 and FIG. 6B shows that the VLPs comprise HA5 Vietnam and M1.

FIG. 7, panels A and B, depict electron micrographs of exemplarypolyvalent VLPs. FIG. 7A shows negative straining of polyvalent VLPs anddepicts polymorphic influenza-virus like structures. Arrows point to HAspikes. FIG. 7B show results of dual immuno-gold labeling of VLPs withanti-1918 HA (10 nm gold particles) and anti-HA5 (5 nm gold particles)antibodies.

FIG. 8 is a graph depicting ELISA assay results. When an anti-H3N2antibody was used in the ELISA, no signal was detected.

FIG. 9 depicts a hemagglutination inhibition (HAI) assay of polyvalentVLPs incubated with both anti-1918 HA and anti-H5 HA Vietnam antibodies.

FIG. 10 is a schematic depicting Western blot/Immunoprecipitationanalysis demonstrating that two different HA molecules (HA5 and HA1918)are expressed on the same surface of the same polyvalent VLP. See,Example, 1. Both blots show markers in the left lane and dual HA VLP inthe right lane. The blots show that the VLPs contain both HA 1918 (leftblot), HA5 Vietnam (right blot) on their surface, in addition to M1(right blot) and M2 (right blot) proteins.

FIG. 11 depicts the nucleotide (SEQ ID NOs:2 and 3) and amino acidsequence (SEQ ID NO:4) of amino terminus of A/Udorn/72 (H3N2)Neuraminidase (NA) protein, including the cytoplasmic tail andtransmembrane domains. The NA cytoplasmic tail contains six N-terminalresidues (MNPNQK, shown in bold) which are identical sequence almost allnine known NA subtypes. The transmembrane domain is underlined.

FIG. 12 depicts the (SEQ ID NOs:5 and 6) nucleotide and amino acidsequence (SEQ ID NO:7) of amino terminus of A/Udorn/72 hemagglutinin(HA) protein, including the cytoplasmic tail and transmembrane domains.The HA cytoplasmic tail contains 10-12 residues (QKGNIRCNICI, shown inbold) which are highly conserved between influenza strains. Thetransmembrane domain is underlined and three residues of the ectodomain(YKD) are shown in lower case.

FIG. 13 depicts the nucleotide (SEQ ID NO:8) and amino acid sequence(SEQ ID NO:9) of amino terminus of influenza A/PR/8/34 (H1N1)Neuraminidase (NA) protein, including the cytoplasmic tail andtransmembrane domains. The NA cytoplasmic tail contains six N-terminalresidues (MNPNQK, shown in bold) which are identical sequence almost allnine known NA subtypes. The transmembrane domain is underlined.

FIG. 14 depicts the nucleotide (SEQ ID NO:10) and amino acid sequence(SEQ ID NO:11) of amino terminus of A/PR/8/34 hemagglutinin (HA)protein, including the cytoplasmic tail and transmembrane domains. TheHA cytoplasmic tail (SNGSLQCRICI) is shown in bold and the transmembranedomain is underlined.

DETAILED DESCRIPTION

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of chemistry, biochemistry, molecularbiology, immunology and pharmacology, within the skill of the art. Suchtechniques are explained fully in the literature. See, e.g., Remington'sPharmaceutical Sciences, 18th Edition (Easton, Pa.: Mack PublishingCompany, 1990); Methods In Enzymology (S. Colowick and N. Kaplan, eds.,Academic Press, Inc.); and Handbook of Experimental Immunology, Vols.I-IV (D. M. Weir and C. C. Blackwell, eds., 1986, Blackwell ScientificPublications); Sambrook, et al., Molecular Cloning: A Laboratory Manual(2nd Edition, 1989); Short Protocols in Molecular Biology, 4th ed.(Ausubel et al. eds., 1999, John Wiley & Sons); Molecular BiologyTechniques: An Intensive Laboratory Course, (Ream et al., eds., 1998,Academic Press); PCR (Introduction to Biotechniques Series), 2nd ed.(Newton & Graham eds., 1997, Springer Verlag); Fundamental Virology,Second Edition (Fields & Knipe eds., 1991, Raven Press, New York).

All publications, patents and patent applications cited herein arehereby incorporated by reference in their entirety.

As used in this specification and the appended claims, the singularforms “a,” “an” and “the” include plural references unless the contentclearly dictates otherwise. Thus, for example, reference to “a VLP”includes a mixture of two or more such VLPs.

DEFINITIONS

As used herein, the terms “sub-viral particle” “virus-like particle” or“VLP” refer to a nonreplicating, viral shell, preferably derived frominfluenza virus proteins. VLPs are generally composed of one or moreviral proteins, such as, but not limited to those proteins referred toas capsid, coat, shell, surface and/or envelope proteins, orparticle-forming polypeptides derived from these proteins. VLPs can formspontaneously upon recombinant expression of the protein in anappropriate expression system. Methods for producing particular VLPs areknown in the art and discussed more fully below. The presence of VLPsfollowing recombinant expression of viral proteins can be detected usingconventional techniques known in the art, such as by electronmicroscopy, biophysical characterization, and the like. See, e.g., Bakeret al., Biophys. J. (1991) 60:1445-1456; Hagensee et al., J. Virol.(1994) 68:4503-4505. For example, VLPs can be isolated by densitygradient centrifugation and/or identified by characteristic densitybanding (e.g., Examples). Alternatively, cryoelectron microscopy can beperformed on vitrified aqueous samples of the VLP preparation inquestion, and images recorded under appropriate exposure conditions.

By “particle-forming polypeptide” derived from a particular viralprotein is meant a full-length or near full-length viral protein, aswell as a fragment thereof, or a viral protein with internal deletions,which has the ability to form VLPs under conditions that favor VLPformation. Accordingly, the polypeptide may comprise the full-lengthsequence, fragments, truncated and partial sequences, as well as analogsand precursor forms of the reference molecule. The term thereforeintends deletions, additions and substitutions to the sequence, so longas the polypeptide retains the ability to form a VLP. Thus, the termincludes natural variations of the specified polypeptide sincevariations in coat proteins often occur between viral isolates. The termalso includes deletions, additions and substitutions that do notnaturally occur in the reference protein, so long as the protein retainsthe ability to form a VLP. Preferred substitutions are those which areconservative in nature, i.e., those substitutions that take place withina family of amino acids that are related in their side chains.Specifically, amino acids are generally divided into four families: (1)acidic—aspartate and glutamate; (2) basic—lysine, arginine, histidine;(3) non-polar—alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan; and (4) uncharged polar—glycine,asparagine, glutamine, cysteine, serine threonine, tyrosine.Phenylalanine, tryptophan, and tyrosine are sometimes classified asaromatic amino acids.

An “antigen” refers to a molecule containing one or more epitopes(either linear, conformational or both) that will stimulate a host'simmune-system to make a humoral and/or cellular antigen-specificresponse. The term is used interchangeably with the term “immunogen.”Normally, a B-cell epitope will include at least about 5 amino acids butcan be as small as 3-4 amino acids. A T-cell epitope, such as a CTLepitope, will include at least about 7-9 amino acids, and a helperT-cell epitope at least about 12-20 amino acids. Normally, an epitopewill include between about 7 and 15 amino acids, such as, 9, 10, 12 or15 amino acids. The term includes polypeptides which includemodifications, such as deletions, additions and substitutions (generallyconservative in nature) as compared to a native sequence, so long as theprotein maintains the ability to elicit an immunological response, asdefined herein. These modifications may be deliberate, as throughsite-directed mutagenesis, or may be accidental, such as throughmutations of hosts which produce the antigens.

An “immunological response” to an antigen or composition is thedevelopment in a subject of a humoral and/or a cellular immune responseto an antigen present in the composition of interest. For purposes ofthe present disclosure, a “humoral immune response” refers to an immuneresponse mediated by antibody molecules, while a “cellular immuneresponse” is one mediated by T-lymphocytes and/or other white bloodcells. One important aspect of cellular immunity involves anantigen-specific response by cytolytic T-cells (“CTL”s). CTLs havespecificity for peptide antigens that are presented in association withproteins encoded by the major histocompatibility complex (MHC) andexpressed on the surfaces of cells. CTLs help induce and promote thedestruction of intracellular microbes, or the lysis of cells infectedwith such microbes. Another aspect of cellular immunity involves anantigen-specific response by helper T-cells. Helper T-cells act to helpstimulate the function, and focus the activity of, nonspecific effectorcells against cells displaying peptide antigens in association with MHCmolecules on their surface. A “cellular immune response” also refers tothe production of cytokines, chemokines and other such moleculesproduced by activated T-cells and/or other white blood cells, includingthose derived from CD4+ and CD8+ T-cells. Hence, an immunologicalresponse may include one or more of the following effects: theproduction of antibodies by B-cells; and/or the activation of suppressorT-cells and/or γΔ T-cells directed specifically to an antigen orantigens present in the composition or vaccine of interest. Theseresponses may serve to neutralize infectivity, and/or mediateantibody-complement, or antibody dependent cell cytotoxicity (ADCC) toprovide protection to an immunized host. Such responses can bedetermined using standard immunoassays and neutralization assays, wellknown in the art.

An “immunogenic composition” is a composition that comprises anantigenic molecule where administration of the composition to a subjectresults in the development in the subject of a humoral and/or a cellularimmune response to the antigenic molecule of interest.

“Substantially purified” general refers to isolation of a substance(compound, polynucleotide, protein, polypeptide, polypeptidecomposition) such that the substance comprises the majority percent ofthe sample in which it resides. Typically in a sample a substantiallypurified component comprises 50%, preferably 80%-85%, more preferably90-95% of the sample. Techniques for purifying polynucleotides andpolypeptides of interest are well-known in the art and include, forexample, ion-exchange chromatography, affinity chromatography andsedimentation according to density.

A “coding sequence” or a sequence which “encodes” a selectedpolypeptide, is a nucleic acid molecule which is transcribed (in thecase of DNA) and translated (in the case of mRNA) into a polypeptide invivo when placed under the control of appropriate regulatory sequences(or “control elements”). The boundaries of the coding sequence aredetermined by a start codon at the 5′ (amino) terminus and a translationstop codon at the 3′ (carboxy) terminus. A coding sequence can include,but is not limited to, cDNA from viral, prokaryotic or eukaryotic mRNA,genomic DNA sequences from viral or prokaryotic DNA, and even syntheticDNA sequences. A transcription termination sequence may be located 3′ tothe coding sequence.

Typical “control elements”, include, but are not limited to,transcription promoters, transcription enhancer elements, transcriptiontermination signals, polyadenylation sequences (located 3′ to thetranslation stop codon), sequences for optimization of initiation oftranslation (located 5′ to the coding sequence), and translationtermination sequences, see e.g., McCaughan et al. (1995) PNAS USA92:5431-5435; Kochetov et al (1998) FEBS Letts. 440:351-355.

A “nucleic acid” molecule can include, but is not limited to,prokaryotic sequences, eukaryotic mRNA, cDNA from eukaryotic mRNA,genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and evensynthetic DNA sequences. The term also captures sequences that includeany of the known base analogs of DNA and RNA.

“Operably linked” refers to an arrangement of elements wherein thecomponents so described are configured so as to perform their usualfunction. Thus, a given promoter operably linked to a coding sequence iscapable of effecting the expression of the coding sequence when active.The promoter need not be contiguous with the coding sequence, so long asit functions to direct the expression thereof. Thus, for example,intervening untranslated yet transcribed sequences can be presentbetween the promoter sequence and the coding sequence and the promotersequence can still be considered “operably linked” to the codingsequence.

“Recombinant” as used herein to describe a nucleic acid molecule means apolynucleotide of genomic, cDNA, semisynthetic, or synthetic originwhich, by virtue of its origin or manipulation: (1) is not associatedwith all or a portion of the polynucleotide with which it is associatedin nature; and/or (2) is linked to a polynucleotide other than that towhich it is linked in nature. The term “recombinant” as used withrespect to a protein or polypeptide means a polypeptide produced byexpression of a recombinant polynucleotide. “Recombinant host cells,”“host cells,” “cells,” “cell lines,” “cell cultures,” and other suchterms denoting prokaryotic microorganisms or eukaryotic cell linescultured as unicellular entities, are used interchangeably, and refer tocells which can be, or have been, used as recipients for recombinantvectors or other transfer DNA, and include the progeny of the originalcell which has been transfected. It is understood that the progeny of asingle parental cell may not necessarily be completely identical inmorphology or in genomic or total DNA complement to the original parent,due to accidental or deliberate mutation. Progeny of the parental cellwhich are sufficiently similar to the parent to be characterized by therelevant property, such as the presence of a nucleotide sequenceencoding a desired peptide, are included in the progeny intended by thisdefinition, and are covered by the above terms.

Techniques for determining amino acid sequence “similarity” are wellknown in the art. In general, “similarity” means the exact amino acid toamino acid comparison of two or more polypeptides at the appropriateplace, where amino acids are identical or possess similar chemicaland/or physical properties such as charge or hydrophobicity. A so-termed“percent similarity” then can be determined between the comparedpolypeptide sequences. Techniques for determining nucleic acid and aminoacid sequence identity also are well known in the art and includedetermining the nucleotide sequence of the mRNA for that gene (usuallyvia a cDNA intermediate) and determining the amino acid sequence encodedthereby, and comparing this to a second amino acid sequence. In general,“identity” refers to an exact nucleotide to nucleotide or amino acid toamino acid correspondence of two polynucleotides or polypeptidesequences, respectively.

Two or more polynucleotide sequences can be compared by determiningtheir “percent identity.” Two or more amino acid sequences likewise canbe compared by determining their “percent identity.” The percentidentity of two sequences, whether nucleic acid or peptide sequences, isgenerally described as the number of exact matches between two alignedsequences divided by the length of the shorter sequence and multipliedby 100. An approximate alignment for nucleic acid sequences is providedby the local homology algorithm of Smith and Waterman, Advances inApplied Mathematics 2:482-489 (1981). This algorithm can be extended touse with peptide sequences using the scoring matrix developed byDayhoff, Atlas of Protein Sequences and Structure, M. O. Dayhoff ed., 5suppl. 3:353-358, National Biomedical Research Foundation, Washington,D.C., USA, and normalized by Gribskov, Nucl. Acids Res. 14(6):6745-6763(1986). Suitable programs for calculating the percent identity orsimilarity between sequences are generally known in the art.

A “vector” is capable of transferring gene sequences to target cells(e.g., bacterial plasmid vectors, viral vectors, non-viral vectors,particulate carriers, and liposomes). Typically, “vector construct,”“expression vector,” and “gene transfer vector,” mean any nucleic acidconstruct capable of directing the expression of one or more sequencesof interest in a host cell. Thus, the term includes cloning andexpression vehicles, as well as viral vectors. The term is usedinterchangeable with the terms “nucleic acid expression vector” and“expression cassette.”

By “subject” is meant any member of the subphylum chordata, including,without limitation, humans and other primates, including non-humanprimates such as chimpanzees and other apes and monkey species; farmanimals such as cattle, sheep, pigs, goats and horses; domestic mammalssuch as dogs and cats; laboratory animals including rodents such asmice, rats and guinea pigs; birds, including domestic, wild and gamebirds such as chickens, turkeys and other gallinaceous birds, ducks,geese, and the like. The term does not denote a particular age. Thus,both adult and newborn individuals are intended to be covered. Thesystem described above is intended for use in any of the abovevertebrate species, since the immune systems of all of these vertebratesoperate similarly.

By “pharmaceutically acceptable” or “pharmacologically acceptable” ismeant a material which is not biologically or otherwise undesirable,i.e., the material may be administered to an individual in a formulationor composition without causing any undesirable biological effects orinteracting in a deleterious manner with any of the components of thecomposition in which it is contained.

As used herein, “treatment” refers to any of (i) the prevention ofinfection or reinfection, as in a traditional vaccine, (ii) thereduction or elimination of symptoms, and (iii) the substantial orcomplete elimination of the pathogen in question. Treatment may beeffected prophylactically (prior to infection) or therapeutically(following infection).

General Overview

Described herein are polyvalent VLPs that can be used to protect humansfrom the predominantly circulating epidemic strains and may be readilyreformulated to accommodate new antigenic drifts. The emergence of novelinfluenza viruses may result in strains completely unknown to the humanimmune system and, if transmissible, may result in a pandemic. Forexample, the avian influenza H5N1 virus has caused deadly outbreaks inavian and mammalian species (including humans) since 1997. This virushas evolved into two distinct phylogenetic clades—clade 1 virusescirculating in Cambodia, Thailand and Vietnam and clade 2 viruses whichcirculated in birds in China and Indonesia during 2003-2004, and spreadwest into Europe, Middle East and Africa, have been responsible forhuman infections during 2005 and 2006. However, because these viruses donot cross-neutralize, each one is independently able to cause severe andfatal cases of influenza in humans. Given the uncertainty as to whichone of these viruses may ultimately acquire the ability to efficientlytransmit among humans creates the dilemma as to which virus/es (Vietnam,clade 1 or Indonesia clade 2) should be included in conventional,egg-produced vaccines.

Thus, the polyvalent VLPs described herein allow for protection ofmultiple influenza strains. These polyvalent virus-like particle (VLP)vaccines carry on their surfaces HA and/or NA molecules in anycombination (e.g., representative of clade 1 and 2 of the H5N1 virus).

The polyvalent influenza virus-like particles (VLPs) described hereinincrease antigenic coverage, reduce development and manufacturing times,as well as costs. Production of our vaccine involves the use of cellbased systems which yields safe (non-infectious) virus-like particlesthat maintain native antigenic epitopes because chemical inactivation isnot required. The lack of need for chemical inactivation will alsoreduce production time and cost. In addition, influenza VLPs can beadministered intranasally. See, e.g., Latham & Galarza (2001) J. Virol.75(13):6154-6165; Galarza et al. (2005) Viral. Immunol. 18(1):244-51;and U.S. Patent Publication 200550186621.

Advantages of the present disclosure include (i) rapid and flexiblecloning methods for influenza vaccine development, (ii) cell-basedinfluenza vaccine production system, (ii) non-infectious (safe)virus-like particles vaccines that do not require inactivation, (iii)two or more antigenically distinct component in one preparation, and(iv) intranasal immunization.

Virus-Like Particles

When sequences encoding influenza proteins are expressed in eukaryotic,the proteins have been shown to self-assemble into noninfectiousvirus-like particles (VLP). See, Latham & Galarza (2001) J. Virol.75(13):6154-6165; Galarza et al. (2005) Viral. Immunol. 18(1):244-51;and U.S. Patent Publications 200550186621 and 20060263804.

The present disclosure relates to the assembly and release of polyvalentinfluenza VLPs from the plasma membrane of eukaryotic cells, which VLPscarry on their surfaces two or more antigenically distinct surfaceglycoproteins. This polyvalent VLP, alone or in combination with one ormore adjuvants, stimulates an immune response that protects againstinfection with any of the antigenically distinct influenza viruses orviral pathogens from which the surface antigens were derived.

The polyvalent VLP (also called sub-viral structure vaccine (SVSV)) iscomposed of viral proteins produced from naturally occurring and/ormutated nucleic acid sequences of genes coding for matrix protein M1 andtwo or more antigenically distinct surface glycoproteins derived fromdifferent viruses and containing engineered unique structural motifs foroptimal molecular contacts with the oligomerized matrix protein M1scaffold. The matrix protein M1 is a universal component for theformation of all possible polyvalent sub-viral structure vaccinecombinations. An M2 protein is optionally included may or may not bepart of the sub-viral structure and its incorporation depends on thedesired antigenic composition of the final vaccine product. Whenpresent, the M2 protein may be modified as described herein.

Antigenically distinct glycoproteins derived from the same or differentfamilies of enveloped viruses can be selected for incorporation onto thesurface of the vaccine. The incorporation of antigenically distinctviral glycoproteins into the same vaccine particle can be facilitated byreplacing the cytoplasmic tail and transmembrane amino acid sequenceswith those from a common glycoprotein via alterations in the nucleicacids coding for these proteins. This approach allows for the design ofa large number of possible polyvalent sub-viral vaccine combinations.

1. Influenza Polypeptide-Encoding Sequences

The VLPs produced as described herein are conveniently prepared usingstandard recombinant techniques. Polynucleotides encoding the influenzaproteins are introduced into a host cell and, when the influenzaproteins are expressed in the cell, they assembly into VLPs.

Polynucleotide sequences coding for molecules (structural and/or antigenpolypeptides) that form and/or incorporate into the VLPs can be obtainedusing recombinant methods, such as by screening cDNA and genomiclibraries from cells expressing the gene, or by deriving the gene from avector known to include the same. For example, plasmids which containsequences that encode naturally occurring or altered cellular productsmay be obtained from a depository such as the A.T.C.C., or fromcommercial sources. Plasmids containing the nucleotide sequences ofinterest can be digested with appropriate restriction enzymes, and DNAfragments containing the nucleotide sequences can be inserted into agene transfer vector using standard molecular biology techniques.

Alternatively, cDNA sequences may be obtained from cells which expressor contain the sequences, using standard techniques, such as phenolextraction and PCR of cDNA or genomic DNA. See, e.g., Sambrook et al.,supra, for a description of techniques used to obtain and isolate DNA.Briefly, mRNA from a cell which expresses the gene of interest can bereverse transcribed with reverse transcriptase using oligo-dT or randomprimers. The single stranded cDNA may then be amplified by PCR (see U.S.Pat. Nos. 4,683,202, 4,683,195 and 4,800,159, see also PCR Technology:Principles and Applications for DNA Amplification, Erlich (ed.),Stockton Press, 1989)) using oligonucleotide primers complementary-tosequences on either side of desired sequences.

The nucleotide sequence of interest can also be produced synthetically,rather than cloned, using a DNA synthesizer (e.g., an Applied BiosystemsModel 392 DNA Synthesizer, available from ABI, Foster City, Calif.). Thenucleotide sequence can be designed with the appropriate codons for theexpression product desired. The complete sequence is assembled fromoverlapping oligonucleotides prepared by standard methods and assembledinto a complete coding sequence. See, e.g., Edge (1981) Nature 292:756;Nambair et al. (1984) Science 223:1299; Jay et al. (1984) J. Biol. Chem.259:6311.

The influenza VLPs described herein are typically formed by expressingsequences encoding M1 and two or more antigenic influenza glycoproteins(HA and NA) in a host cell. The VLPs may optionally comprise M2. Theexpressed proteins self-assemble into VLPs with the antigenicglycoproteins decorating the surface of the VLP.

The sequences can be derived from any two influenza strains, preferablyinfluenza A strains or two different influenza B strains. There arethree types of influenza viruses: A, B, and C. Influenza A viruses arefurther classified by subtype on the basis of the two main surfaceglycoproteins hemagglutinin (HA) and neuraminidase (NA) (e.g., H1N1,H1N2, H3N2, H3N5, etc.). There are 16 known HA subtypes (H1, H2, H3, H4,H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16) and 9 known NAsubtypes (N1, N2, N3, N4, N5, N6, N7, N8, N9). The VLPs described hereincan include any combination of these 16 HA subtypes and/or these 9 NAsubtypes.

As hundreds of strains of influenza viruses have been identified and arecontinually being identified, it will be apparent that glycoproteinsfrom any of these strains can be incorporated into the VLPs describedherein. Examples of strains from which influenza encoding sequences canbe derived include those listed by the World Health Organizations andthe Centers for Disease Control (CDC) (both organizations maintain listsof pathogenic strains which are available on the internet). Non-limitingexamples of such influenza strains include: A/Puerto Rico/8/34 (H1N1),A/Asian/57 (H2N2), A/Hong Kong/68 (H3N2), A/New York/55/04 (H3N2),ANietnam/1203/04 (H5N1)

The polyvalent VLPs described herein incorporate any combination of HAand/or NA antigenic polypeptides from different strains or subtypes.Furthermore, it will be apparent that antigenic drift and antigenicshift occur in flu viruses, resulting in new strains. When a new strainof human influenza virus emerges, antibody protection that may havedeveloped after infection or vaccination with an older strain may notprovide protection against the new strain. Therefore, the disclosurecontemplates use of sequences from both identified and unidentifiedstrains in the formation of polyvalent VLPs. See, also the internet forupdated virus listings, for example the World Health Organization sitereferenced above.

The VLPs described herein may further comprise an influenzanucleoprotein (NP) and/or at least one protein of the polymerasecomplex, i.e., one or more of PB1, PB2 and PA (e.g., PB1, PB2, and PA;PB1 and PB2; PB1 and PA; PB2 and PA). Preferably, the VLPs do notinclude NP and all three proteins of the polymerase complex. Thestructure and function of influenza NP and polymerase complex proteinsis known and described for example on pages 428 to 432 of KubyImmunology, 4^(th) ed. (Goldsby et al. eds.) WH Freeman & Company, NewYork.

The sequences may be naturally occurring sequences or, alternatively,may include modifications, made using standard molecular biologicaltechniques, for example to enhance expression, interaction with otherproteins (e.g., M1), etc. See, Examples.

The polyvalent influenza VLP are typically formed by expressing one ormore influenza matrix proteins and one or more antigenic influenzaglycoproteins (HA and NA).

In certain embodiments, the sequences encode naturally occurring ormodified M1 and/or M2 are selected for use. The sequences encoding thematrix protein(s) may be derived from any influenza virus, for examplefrom the influenza virus strain A/PR/8 (H1N1) or influenza strainA/Udorn/72 (H3N2). Exemplary modifications, with respect to the matrixproteins of A/Udorn/72 are shown in Tables 1 and 2 below.

TABLE 1 M1 Domain Residues* Exemplary Changes NLS 101-105 Deletion,insertion and/or substitution of one or more residues sphericalstructure 41 deletion and/or determinants 95 substitution of 98 residuesand/or one or more 167 surrounding residues 204 205 218 self and 4Deletion, insertion and/or oligomerization 16 substitution of domains 19one or more residues 33 50 51 52 59 66 67 L-domains Self 100-103introduce L-domains as or from other additions or instead viruses ofnative sequences *numbered relative to 252 amino acid sequence ofA/Udorn/72 M1 as shown in GenBank Accession No. ABD79033 M2 DomainResidues* Exemplary Changes L-domains 100-103 from introduce L-domainsas M1 or L additions or instead domains from of native sequences otherviruses Addition of sequences C- or N- addition of sequences encodingterminus encoding immunodulators immunomodulators (e.g., adjuvants)*numbered relative to 96 amino acid sequence of A/Udorn/72 M2 shown inGenBank Accession No. ABD79034

The matrix proteins of various influenza proteins are typically fairlyconserved and one of skill in the art can readily align sequences fromany given strains to determine domains and regions corresponding to thethose set forth above.

Thus, as shown above, in certain embodiments, the M1 protein containsone or more modifications shown in Table 1. For example, the nuclearlocalization signal (NLS), one or more determinants of sphericalstructure (e.g., amino acid residues 41, 95, 98, 167, 204, 205 and 218,numbered relative to A/Udorn/72); one or more regions involved inself-protein interactions and/or oligomerization may be modified; and/orone or more L-domains may be introduced. See, FIG. 2. L-domains arespecific motifs that interact with cellular components which arerecruited for budding and pinching off of the virus or virus-likeparticles from the cell membrane. L-domains of certain viruses are ableto function in a position independent manner and some of them arefunctionally interchangeable among different viruses. Hui et al. (2003)J. Virol. 77:7078-7092; Hui et. al. (2006), J. Virol. 80:2291-2308;Freed, E. O. (2002) J. Virol. 76:4679-4687

During viral infection, a significant proportion of the synthesizedmatrix protein translocates to the nucleus of the infected cells whereit participates in ribonucleoprotein (RNP) translocation to thecytoplasm and transport to the cell periphery for virus assembly andbudding. A smaller fraction of produced matrix protein directlyassociates with the plasma membrane and is the driving force in virusassembly and budding. When matrix protein is expressed in eukaryoticcells alone or in combination with other viral structural proteins itdemonstrates a similar distribution pattern as in infected cells.Modification of the nuclear localization signal alters this distributionpattern by diverting most of the matrix protein to the cell peripherywhere it associates with the inner leaflet of the plasma membraneenhancing protein-protein interaction and self oligomerization leadingto greater frequency of sub-viral particle assembly and release. Thus,such changes may enhance the efficiency of sub-viral structure assemblyand release, therefore increasing vaccine production yield.

Sequences encoding HA and/or NA glycoproteins are also provided and areincorporate into the influenza VLPs such that they are expressed on thesurface. The sequences encoding the glycoprotein(s) may be naturallyoccurring or modified (e.g., by deletions, additions and/orsubstitutions) (see, Examples).

In certain embodiments, the sequences encode chimeric polypeptides, forexample chimeric glycoproteins (HA and/or NA) or hybridmatrix-immunomodulatory polypeptides are used in constructing a VLP. Itwill be apparent that all or parts of the polypeptides may be replacedwith sequences from other viruses and/or sequences from other influenzastrains. In a preferred embodiment, the sequences encoding the HA and/orNA glycoproteins are chimeric in that they include heterologoussequences encoding the transmembrane and/or cytoplasmic tail domains.See, FIGS. 4 and 11-14. For example, the transmembrane domain andcytoplasmic tail of both HA5 and HA 1918 may be replaced by thehomologous domains derived from influenza A/Udorn/72 or A/PR/8/34. TheHA molecule is a type I glycoprotein, thus the trans-membrane andcytoplasmic tail exchanged are located at the carboxyl-terminal (COOH₂)end of the molecule. In the case of NA, a type II glycoprotein, theexchanged domains are located at the amino-terminal (NH₂) end of themolecule. These exchanges enhance the interaction of the surfaceglycoproteins with the scaffold formed by the matrix protein M1, whichis also derived from either influenza A/Udorn/72 or A/PR/8/34 virus andunderlies the membrane of the sub-viral structure. See, FIGS. 11 to 14

Preferably, the influenza sequences employed to form influenza VLPsexhibit between about 60% to 80% (or any value therebetween including61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78% and 79%) sequence identity to a naturally occurringinfluenza polynucleotide sequence and more preferably the sequencesexhibit between about 80% and 100% (or any value therebetween including81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% and 99%) sequence identity to a naturally occurringinfluenza polynucleotide sequence.

Any of the sequences described herein may further include additionalsequences. For example, to further to enhance vaccine potency, hybridmolecules are expressed and incorporated into the sub-viral structure.These hybrid molecules are generated by linking, at the DNA level, thesequences coding for the matrix protein genes with sequences coding foran adjuvant or immuno-regulatory moiety. During sub-viral structureformation, these chimeric proteins are incorporated into or onto theparticle depending on whether M1 or optional M2 carries the adjuvantmolecule. The incorporation of one or more polypeptide immunomodulatorypolypeptides (e.g., adjuvants describe in detail below) into thesequences described herein into the VLP may enhance potency andtherefore reduces the amount of antigen required for stimulating aprotective immune response. Alternatively, as described below, one ormore additional molecules (polypeptide or small molecules) may beincluded in the VLP-containing compositions after production of the VLPfrom the sequences described herein.

These sub-viral structures do not contain infectious viral nucleic acidsand they are not infectious eliminating the need for chemicalinactivation. Absence of chemical treatment preserves native epitopesand protein conformations enhancing the immunogenic characteristics ofthe vaccine.

The sequences described herein can be operably linked to each other inany combination. For example, one or more sequences may be expressedfrom the same promoter and/or from different promoters. As describedbelow, sequences may be included on one or more vectors.

The polyvalent VLPs described herein comprise multiple copies ofantigenic flu proteins (e.g., HA and/or NA) from two or more differentinfluenza strains.

2. Expression Vectors

Once the constructs comprising the sequences encoding the influenzapolypeptides desired to be incorporated into the VLP have beensynthesized, they can be cloned into any suitable vector or replicon forexpression. Numerous cloning vectors are known to those of skill in theart, and one having ordinary skill in the art can readily selectappropriate vectors and control elements for any given host cell type inview of the teachings of the present specification and information knownin the art about expression. See, generally, Ausubel et al, supra orSambrook et al, supra.

Non-limiting examples of vectors that can be used to express sequencesthat assembly into VLPs as described herein include viral-based vectors(e.g., retrovirus, adenovirus, adeno-associated virus, lentivirus),baculovirus vectors (see, Examples), plasmid vectors, non-viral vectors,mammalians vectors, mammalian artificial chromosomes (e.g., liposomes,particulate carriers, etc.) and combinations thereof.

The expression vector(s) typically contain(s) coding sequences andexpression control elements which allow expression of the coding regionsin a suitable host. The control elements generally include a promoter,translation initiation codon, and translation and transcriptiontermination sequences, and an insertion site for introducing the insertinto the vector. Translational control elements have been reviewed by M.Kozak (e.g., Kozak, M., Mamm. Genome 7(8):563-574, 1996; Kozak, M.,Biochimie 76(9):815-821, 1994; Kozak, M., J Cell Biot 108(2):229-241,1989; Kozak, M., and Shatkin, A. J., Methods Enzymol 60:360-375, 1979).

For example, typical promoters for mammalian cell expression include theSV40 early promoter, a CMV promoter such as the CMV immediate earlypromoter (a CMV promoter can include intron A), RSV, HIV-LTR, the mousemammary tumor virus LTR promoter (MMLV-LTR), FIV-LTR, the adenovirusmajor late promoter (Ad MLP), and the herpes simplex virus promoter,among others. Other nonviral promoters, such as a promoter derived fromthe murine metallothionein gene, will also find use for mammalianexpression. Typically, transcription termination and polyadenylationsequences will also be present, located 3′ to the translation stopcodon. Preferably, a sequence for optimization of initiation oftranslation, located 5′ to the coding sequence, is also present.Examples of transcription terminator/polyadenylation signals includethose derived from SV40, as described in Sambrook, et al., supra, aswell as a bovine growth hormone terminator sequence. Introns, containingsplice donor and acceptor sites, may also be designed into theconstructs as described herein (Chapman et al., Nuc. Acids Res. (1991)19:3979-3986).

Enhancer elements may also be used herein to increase expression levelsof the mammalian constructs. Examples include the SV40 early geneenhancer, as described in Dijkema et al., EMBO J. (1985) 4:761, theenhancer/promoter derived from the long terminal repeat (LTR) of theRous Sarcoma Virus, as described in Gorman et al., Proc. Natl. Acad.Sci. USA (1982b) 79:6777 and elements derived from human CMV, asdescribed in Boshart et al., Cell (1985) 41:521, such as elementsincluded in the CMV intron A sequence (Chapman et al., Nuc. Acids Res.(1991) 19:3979-3986).

It will be apparent that one or more vectors may contain one or moresequences encoding proteins to be incorporated into the VLP. Forexample, a single vector may carry sequences encoding all the proteinsfound in the VLP. Alternatively, multiple vectors may be used (e.g.,multiple constructs, each encoding a single polypeptide-encodingsequence or multiple constructs, each encoding one or morepolypeptide-encoding sequences). In embodiments in which a single vectorcomprises multiple polypeptide-encoding sequences, the sequences may beoperably linked to the same or different transcriptional controlelements (e.g., promoters) within the same vector.

In addition, one or more sequences encoding non-influenza proteins maybe expressed and incorporated into the VLP, including, but not limitedto, sequences comprising and/or encoding immunomodulatory molecules(e.g., adjuvants described below), for example, immunomodulatingoligonucleotides (e.g., CpGs), cytokines, detoxified bacterial toxinsand the like.

3. VLP Production

As noted above, influenza proteins expressed in a eukaryotic host cellhave been shown to self-assemble into noninfectious virus-like particles(VLP). Accordingly, the sequences and/or vectors described herein arethen used to transform an appropriate host cell. The construct(s)encoding the proteins that form the VLPs described herein provideefficient means for the production of influenza VLPs using a variety ofdifferent cell types, including, but not limited to, insect, fungal(yeast) and mammalian cells.

Preferably, the sub-viral structure vaccines are produced in eukaryoticcells following transfection, establishment of continuous cell lines(using standard protocols) and/or infection with DNA constructs thatcarry the influenza genes of interest as known to one skilled in theart. The level of expression of the proteins required for sub-viralstructure formation is maximized by sequence optimization of theeukaryotic or viral promoters that drive transcription of the selectedgenes. The sub-viral structure vaccine is released into the culturemedium, from where it is purified and subsequently formulated as avaccine. The sub-viral structures are not infectious and thereforeinactivation of the VLP is not required as it is for some killed viralvaccines

The ability of influenza polypeptides expressed from sequences asdescribed herein to self-assemble into VLPs with antigenic glycoproteinspresented on the surface allows these VLPs to be produced in many hostcell by co-introduction of the desired sequences. The sequence(s) (e.g.,in one or more expression vectors) may be stably and/or transientlyintegrated in various combinations into a host cell.

Suitable host cells include, but are not limited to, bacterial,mammalian, baculovirus/insect, yeast, plant and Xenopus cells.

For example, a number of mammalian cell lines are known in the art andinclude primary cells as well as immortalized cell lines available fromthe American Type Culture Collection (A.T.C.C.), such as, but notlimited to, BHK, VERO, MRC-5, WI-38, HT1080, 293, 293T, RD, COS-7, CHO,Jurkat, HUT, SUPT, C8166, MOLT4/clone8, MT-2, MT-4, H9, PM1, CEM,myeloma cells (e.g., SB20 cells) and CEMX174 (such cell lines areavailable, for example, from the A.T.C.C.).

Similarly, bacterial hosts such as E. coli, Bacillus subtilis, andStreptococcus spp., will find use with the present expressionconstructs.

Yeast hosts useful in the present disclosure include inter alia,Saccharomyces cerevisiae, Candida albicans, Candida maltosa, Hansenulapolymorpha, Kluyveromyces fragilis, Kluyveromyces lactis, Pichiaguillerimondii, Pichia pastoris, Schizosaccharomyces pombe and Yarrowialipolytica. Fungal hosts include, for example, Aspergillus.

Insect cells for use with baculovirus expression vectors include, interalia, Aedes aegypti, Autographa californica, Bombyx mori, Drosophilamelanogaster, Spodoptera frugiperda, and Trichoplusia ni. See, Latham &Galarza (2001) J. Virol. 75(13):6154-6165; Galarza et al. (2005) Viral.Immunol. 18(1):244-51; and U.S. Patent Publications 200550186621 and20060263804.

Cell lines expressing one or more of the sequences described above canreadily be generated given the disclosure provided herein by stablyintegrating one or more expression vector constructs encoding theinfluenza proteins of the VLP. The promoter regulating expression of thestably integrated influenza sequences (s) may be constitutive orinducible. Thus, a cell line can be generated in which one or more bothof the matrix proteins are stably integrated such that, uponintroduction of the influenza glycoprotein-encoding sequences describedherein (e.g., chimeric glycoproteins) into a host cell and expression ofthe influenza proteins encoded by the polynucleotides, nonreplicatinginfluenza viral particles that present antigenic glycoproteins areformed.

In certain embodiments, a mammalian cell line that stably expressed twoor more antigenically distinct influenza glycoproteins is generated.Sequences encoding M1, M2 and/or additional glycoproteins (e.g., fromthe same or different virus strains) can be introduced into such a cellline to produce VLPs as described herein. Alternatively, a cell linethat stably produces an influenza M1 protein (and, optionally, M2) canbe generated and sequences encoding the glycoprotein(s) from theselected influenza strain introduced into the cell line, resulting inproduction of VLPs presenting the desired antigenic glycoproteins.

The parent cell line from which an influenza VLP-producer cell line isderived can be selected from any cell described above, including forexample, mammalian, insect, yeast, bacterial cell lines. In a preferredembodiment, the cell line is a mammalian cell line (e.g., 293, RD,COS-7, CHO, BHK, MDCK, MDBK, MRC-5, VERO, HT1080, and myeloma cells).Production of influenza VLPs using mammalian cells provides (i) VLPformation; (ii) correct post translation modifications (glycosylation,palmitylation) and budding; (iii) absence of non-mammalian cellcontaminants and (iv) ease of purification.

In addition to creating cell lines, influenza-encoding sequences mayalso be transiently expressed in host cells. Suitable recombinantexpression host cell systems include, but are not limited to, bacterial,mammalian, baculovirus/insect, vaccinia, Semliki Forest virus (SFV),Alphaviruses (such as, Sindbis, Venezuelan Equine Encephalitis (VEE)),mammalian, yeast and Xenopus expression systems, well known in the art.Particularly preferred expression systems are mammalian cell lines,vaccinia, Sindbis, insect and yeast systems.

Many suitable expression systems are commercially available, including,for example, the following: baculovirus expression (Reilly, P. R., etal., BACULOVIRUS EXPRESSION VECTORS: A LABORATORY MANUAL (1992); Beames,et al., Biotechniques 11:378 (1991); Pharmingen; Clontech, Palo Alto,Calif.)), vaccinia expression systems (Earl, P. L., et al., “Expressionof proteins in mammalian cells using vaccinia” In Current Protocols inMolecular Biology (F. M. Ausubel, et al. Eds.), Greene PublishingAssociates & Wiley Interscience, New York (1991); Moss, B., et al., U.S.Pat. No. 5,135,855, issued Aug. 4, 1992), expression in bacteria(Ausubel, F. M., et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, JohnWiley and Sons, Inc., Media PA; Clontech), expression in yeast(Rosenberg, S. and Tekamp-Olson, P., U.S. Pat. No. RE35,749, issued,Mar. 17, 1998, herein incorporated by reference; Shuster, J. R., U.S.Pat. No. 5,629,203, issued May 13, 1997, herein incorporated byreference; Gellissen, G., et al., Antonie Van Leeuwenhoek, 62(1-2):79-93(1992); Romanos, M. A., et al., Yeast 8(6):423-488 (1992); Goeddel, D.V., Methods in Enzymology 185 (1990); Guthrie, C., and G. R. Fink,Methods in Enzymology 194 (1991)), expression in mammalian cells(Clontech; Gibco-BRL, Ground Island, N.Y.; e.g., Chinese hamster ovary(CHO) cell lines (Haynes, J., et al., Nuc. Acid. Res. 11:687-706 (1983);1983, Lau, Y. F., et al., Mol. Cell. Biol. 4:1469-1475 (1984); Kaufman,R. J., “Selection and coamplification of heterologous genes in mammaliancells,” in Methods in Enzymology, vol. 185, pp 537-566. Academic Press,Inc., San Diego Calif. (1991)), and expression in plant cells (plantcloning vectors, Clontech Laboratories, Inc., Palo-Alto, Calif., andPharmacia LKB Biotechnology, Inc., Pistcataway, N.J.; Hood, E., et al.,J. Bacteriol. 168:1291-1301 (1986); Nagel, R., et al., FEMS Microbiol.Lett. 67:325 (1990); An, et al., “Binary Vectors”, and others in PlantMolecular Biology Manual A3:1-19 (1988); Miki, B. L. A., et al., pp.249-265, and others in Plant DNA Infectious Agents (Hohn, T., et al.,eds.) Springer-Verlag, Wien, Austria, (1987); Plant Molecular Biology:Essential Techniques, P. G. Jones and J. M. Sutton, New York, J. Wiley,1997; Miglani, Gurbachan Dictionary of Plant Genetics and MolecularBiology, New York, Food Products Press, 1998; Henry, R. J., PracticalApplications of Plant Molecular Biology, New York, Chapman & Hall,1997).

When expression vectors containing the altered genes that code for theproteins required for sub-viral structure vaccine formation areintroduced into host cell(s) and subsequently expressed at the necessarylevel, the sub-viral structure vaccine assembles and is then releasedfrom the cell surface into the culture media (FIG. 7).

Depending on the expression system and host selected, the VLPs areproduced by growing host cells transformed by an expression vector underconditions whereby the particle-forming polypeptide is expressed andVLPs can be formed. The selection of the appropriate growth conditionsis within the skill of the art. If the VLPs are formed and retainedintracellularly, the cells are then disrupted, using chemical, physicalor mechanical means, which lyse the cells yet keep the VLPssubstantially intact. Such methods are known to those of skill in theart and are described in, e.g., Protein Purification Applications: APractical Approach, (E. L. V. Harris and S. Angal, Eds., 1990).Alternatively, VLPs may be secreted and harvested from the surroundingculture media.

The particles are then isolated (or substantially purified) usingmethods that preserve the integrity thereof, such as, by densitygradient centrifugation, e.g., sucrose gradients, PEG-precipitation,pelleting, and the like (see, e.g., Kirnbauer et al. J. Virol. (1993)67:6929-6936), as well as standard purification techniques including,e.g., ion exchange and gel filtration chromatography.

Compositions

VLPs produced as described herein can be used to elicit an immuneresponse when administered to a subject. As discussed above, the VLPscan comprise a variety of antigens (e.g., one or more influenza antigensfrom one or more strains or isolates). Purified VLPs can be administeredto a vertebrate subject, usually in the form of vaccine compositions.Combination vaccines may also be used, where such vaccines contain, forexample, other subunit proteins derived from influenza or otherorganisms and/or gene delivery vaccines encoding such antigens.

VLP immune-stimulating (or vaccine) compositions can include variousexcipients, adjuvants, carriers, auxiliary substances, modulatingagents, and the like. The immune stimulating compositions will includean amount of the VLP/antigen sufficient to mount an immunologicalresponse. An appropriate effective amount can be determined by one ofskill in the art. Such an amount will fall in a relatively broad rangethat can be determined through routine trials and will generally be anamount on the order of about 0.1 μg to about 10 (or more) mg, morepreferably about 1 μg to about 300 μg, of VLP/antigen.

Sub-viral structure vaccines are purified from the cell culture mediumand formulated with the appropriate buffers and additives, such as a)preservatives or antibiotics; b) stabilizers, including proteins ororganic compounds; c) adjuvants or immuno-modulators for enhancingpotency and modulating immune responses (humoral and cellular) to thevaccine; or d) molecules that enhance presentation of vaccine antigensto specifics cell of the immune system. This vaccine can be prepared ina freeze-dried (lyophilized) form in order to provide for appropriatestorage and maximize the shelf-life of the preparation. This will allowfor stock piling of vaccine for prolonged periods of time maintainingimmunogenicity, potency and efficacy.

A carrier is optionally present in the compositions described herein.Typically, a carrier is a molecule that does not itself induce theproduction of antibodies harmful to the individual receiving thecomposition. Suitable carriers are typically large, slowly metabolizedmacromolecules such as proteins, polysaccharides, polylactic acids,polyglycollic acids, polymeric amino acids, amino acid copolymers, lipidaggregates (such as oil droplets or liposomes), and inactive virusparticles. Examples of particulate carriers include those derived frompolymethyl methacrylate polymers, as well as microparticles derived frompoly(lactides) and poly(lactide-co-glycolides), known as PLG. See, e.g.,Jeffery et al., Pharm. Res. (1993) 10:362-368; McGee J P, et al., JMicroencapsul. 14(2):197-210, 1997; O'Hagan D T, et al., Vaccine11(2):149-54, 1993. Such carriers are well known to those of ordinaryskill in the art.

Additionally, these carriers may function as immunostimulating agents(“adjuvants”). Exemplary adjuvants include, but are not limited to: (1)aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate,aluminum sulfate, etc.; (2) oil-in-water emulsion formulations (with orwithout other specific immunostimulating agents such as muramyl peptides(see below) or bacterial cell wall components), such as for example (a)MF59 (International Publication No. WO 90/14837), containing 5%Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionally containing variousamounts of MTP-PE (see below), although not required) formulated intosubmicron particles using a microfluidizer such as Model 110Ymicrofluidizer (Microfluidics, Newton, Mass.), (b) SAF, containing 10%Squalane, 0.4% Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP(see below) either microfluidized into a submicron emulsion or vortexedto generate a larger particle size emulsion, and (c) Ribi™ adjuvantsystem (RAS), (Ribi Immunochem, Hamilton, Mont.) containing 2% Squalene,0.2% Tween 80, and one or more bacterial cell wall components from thegroup consisting of monophosphorylipid A (MPL), trehalose dimycolate(TDM), and cell wall skeleton (CWS), preferably MPL+CWS (Detoxu); (3)saponin adjuvants, such as Stimulon™. (Cambridge Bioscience, Worcester,Mass.) may be used or particle generated therefrom such as ISCOMs(immunostimulating complexes); (4) Complete Freunds Adjuvant (CFA) andIncomplete Freunds Adjuvant (IFA); (5) cytokines, such as interleukins(IL-1, IL-2, etc.), macrophage colony stimulating factor (M-CSF), tumornecrosis factor (TNF), beta chemokines (MIP, 1-alpha, 1-beta Rantes,etc.); (6) detoxified mutants of a bacterial ADP-ribosylating toxin suchas a cholera toxin (CT), a pertussis toxin (PT), or an E. coliheat-labile toxin (LT), particularly LT-K63 (where lysine is substitutedfor the wild-type amino acid at position 63) LT-R72 (where arginine issubstituted for the wild-type amino acid at position 72), CT-S109 (whereserine is substituted for the wild-type amino acid at position 109), andPT-K9/G129 (where lysine is substituted for the wild-type amino acid atposition 9 and glycine substituted at position 129) (see, e.g.,International Publication Nos. WO93/13202 and WO92/19265); and (7) othersubstances that act as immunostimulating agents to enhance theeffectiveness of the composition.

Muramyl peptides include, but are not limited to,N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acteyl-normuramyl-L-alanyl-D-isogluatme (nor-MDP),N-acetylmuramyl-L-alanyl-D-isogluatminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-huydroxyphosphoryloxy)-ethylamine(MTP-PE), etc.

Examples of suitable immunomodulatory molecules for use herein includeadjuvants described above and the following: IL-1 and IL-2 (Karupiah etal. (1990) J. Immunology 144:290-298, Weber et al. (1987) J. Exp. Med.166:1716-1733, Gansbacher et al. (1990) J. Exp. Med. 172:1217-1224, andU.S. Pat. No. 4,738,927); IL-3 and IL-4 (Tepper et al. (1989) Cell57:503-512, Golumbek et al. (1991) Science 254:713-716, and U.S. Pat.No. 5,017,691); IL-5 and IL-6 (Brakenhof et al. (1987) J. Immunol.139:4116-4121, and International Publication No. WO 90/06370); IL-7(U.S. Pat. No. 4,965,195); IL-8, IL-9, IL-10, IL-11, IL-12, and IL-13(Cytokine Bulletin, Summer 1994); IL-14 and IL-15; alpha interferon(Finter et al. (1991) Drugs 42:749-765, U.S. Pat. Nos. 4,892,743 and4,966,843, International Publication No. WO 85/02862, Nagata et al.(1980) Nature 284:316-320, Familletti et al. (1981) Methods in Enz.78:387-394, Twu et al. (1989) Proc. Natl. Acad. Sci. USA 86:2046-2050,and Faktor et al. (1990) Oncogene 5:867-872); β-interferon (Seif et al.(1991) J. Virol. 65:664-671); γ-interferons (Watanabe et al. (1989)Proc. Natl. Acad. Sci. USA 86:9456-9460, Gansbacher et al. (1990) CancerResearch 50:7820-7825, Maio et al. (1989) Can. Immunol. Immunother.30:34-42, and U.S. Pat. Nos. 4,762,791 and 4,727,138); G-CSF (U.S. Pat.Nos. 4,999,291 and 4,810,643); GM-CSF (International Publication No. WO85/04188); tumor necrosis factors (TNFs) (Jayaraman et al. (1990) J.Immunology 144:942-951); CD3 (Krissanen et al. (1987) Immunogenetics26:258-266); ICAM-1 (Altman et al. (1989) Nature 338:512-514, Simmons etal. (1988) Nature 331:624-627); ICAM-2, LFA-1, LFA-3 (Wallner et al.(1987) J. Exp. Med. 166:923-932); MHC class I molecules, MHC class IImolecules, B7.1-β2-microglobulin (Parnes et al. (1981) Proc. Natl. Acad.Sci. USA 78:2253-2257); chaperones such as calnexin; and MHC-linkedtransporter proteins or analogs thereof (Powis et al. (1991) Nature354:528-531). Immunomodulatory factors may also be agonists,antagonists, or ligands for these molecules. For example, soluble formsof receptors can often behave as antagonists for these types of factors,as can mutated forms of the factors themselves.

Nucleic acid molecules that encode the above-described substances, aswell as other nucleic acid molecules that are advantageous for usewithin the present invention, may be readily obtained from a variety ofsources, including, for example, depositories such as the American TypeCulture Collection, or from commercial sources such as BritishBio-Technology Limited (Cowley, Oxford England). Representative examplesinclude BBG 12 (containing the GM-CSF gene coding for the mature proteinof 127 amino acids), BBG 6 (which contains sequences encoding gammainterferon), A.T.C.C. Deposit No. 39656 (which contains sequencesencoding TNF), A.T.C.C. Deposit No. 20663 (which contains sequencesencoding alpha-interferon), A.T.C.C. Deposit Nos. 31902, 31902 and 39517(which contain sequences encoding beta-interferon), A.T.C.C. Deposit No.67024 (which contains a sequence which encodes Interleukin-1b), A.T.C.C.Deposit Nos. 39405, 39452, 39516, 39626 and 39673 (which containsequences encoding Interleukin-2), A.T.C.C. Deposit Nos. 59399, 59398,and 67326 (which contain sequences encoding Interleukin-3), A.T.C.C.Deposit No. 57592 (which contains sequences encoding Interleukin-4),A.T.C.C. Deposit Nos. 59394 and 59395 (which contain sequences encodingInterleukin-5), and A.T.C.C. Deposit No. 67153 (which contains sequencesencoding Interleukin-6).

Plasmids encoding one or more of the above-identified polypeptides canbe digested with appropriate restriction enzymes, and DNA fragmentscontaining the particular gene of interest can be inserted into a genetransfer vector (e.g., expression vector as described above) usingstandard molecular biology techniques. (See, e.g., Sambrook et al.,supra, or Ausubel et al. (eds) Current Protocols in Molecular Biology,Greene Publishing and Wiley-Interscience).

Administration

The VLPs and compositions comprising these VLPs can be administered to asubject by any mode of delivery, including, for example, by parenteralinjection (e.g. subcutaneously, intraperitoneally, intravenously,intramuscularly, or to the interstitial space of a tissue), or byrectal, oral (e.g. tablet, spray), vaginal, topical, transdermal (e.g.see WO99/27961) or transcutaneous (e.g. see WO02/074244 andWO02/064162), intranasal (e.g. see WO03/028760), ocular, aural,pulmonary or other mucosal administration. Multiple doses can beadministered by the same or different routes. In a preferred embodiment,the doses are intranasally administered.

The VLPs (and VLP-containing compositions) can be administered prior to,concurrent with, or subsequent to delivery of other vaccines. Also, thesite of VLP administration may be the same or different as other vaccinecompositions that are being administered.

Dosage treatment with the VLP composition may be a single dose scheduleor a multiple dose schedule. A multiple dose schedule is one in which aprimary course of vaccination may be with 1-10 separate doses, followedby other doses given at subsequent time intervals, chosen to maintainand/or reinforce the immune response, for example at 1-4 months for asecond dose, and if needed, a subsequent dose(s) after several months.The dosage regimen will also, at least in part, be determined by thepotency of the modality, the vaccine delivery employed, the need of thesubject and be dependent on the judgment of the practitioner.

All patents, patent applications and publications mentioned herein arehereby incorporated by reference in their entireties.

Although disclosure has been provided in some detail by way ofillustration and example for the purposes of clarity and understanding,it will be apparent to those of skill in the art that various changesand modifications can be practiced without departing from the spirit orscope of the disclosure. Accordingly, the foregoing disclosure andfollowing examples should not be construed as limiting. For instance,although the VLPs disclosed in the Examples include M2, it will beapparent from the above disclosure that M2 is optional and thatinfluenza VLPs as described herein can be formed without M2. See, also,U.S. Patent Publication Nos. 20050186621 and 20060263804.

EXAMPLES Example 1 Polyvalent HA Influenza VLPs

To evaluate the formation of polyvalent influenza VLPs, the HA5 genederived from the human influenza virus ANietnam/1203/2004 (H5N1) wasobtained by RT-PCR from the reassortant virus A/VNH5N1-PR8/CDC-RGreference strain and the gene encoding the 1918 HA (A/SouthCaroline/1/18 (H1N1) was in vitro synthesized following the databasesequence (accession #AF117241). Both genes were cloned into the pAcAB4baculovirus transfer vector (Latham & Galarza (2001) J. Virol.75(13):6154-6165; Galarza et al. (2005) Viral. Immunol. 18(1):244-51;and U.S. Patent Publication 200550186621). This vector contains thegenes encoding the M1 and M2 proteins of the influenza virus A/Udorn/72(H3N2). In this plasmid, the HA5 and M1 genes were under thetranscriptional control of the baculovirus polyhedrin promoter and inopposite orientation, whereas the 1918 HA and the M2 were under thetranscriptional control of the p10 promoter and in opposite orientationto each other (FIG. 5). In this construct, the cytoplasmic tail andtransmembrane domain of both HAs were replaced with the homologousdomains of the HA of the influenza virus A/Udorn/72. Recombinant viruswas generated by co-tansfecting Sf9 insect cells with transfer vectorand a linear baculovirus genomic DNA.

Influenza-virus-like particles (VLPs) were produced by infecting Sf9cells with the quadruple recombinant. Infection was allowed to proceedfor 72 hrs at which time culture supernatant was collected, clarified,concentrated by high speed centrifugation and further purified bygradient centrifugation.

Western Blot/Immunoprecipitation

Western blot analysis of purified polyvalent VLPs with a mousemonoclonal antibody against the 1918 HA (generously provided by Dr.Palese, Mount Sinai School of Medicine) and a rabbit polyclonal antibodyagainst influenza virus ANietnam/1023/04 (H5N1) (generously provided byDr. Donis, Influenza Branch, CDC) demonstrated that both molecules werepresent in the purified VLP preparation. (FIG. 6).

In order to determine whether the two HA molecules were incorporatedonto the surface of the same particle or segregated onto the surface ofdifferent virus-like particles (VLPs), differentialimmuno-precipitation/Western blot analysis was carried out. In thisexperiment, purified VLPs were incubated first with either a mouseanti-1918-HA Mabs or a rabbit anti-avian influenza HA5 (raised against asynthetic peptide) and then precipitated with protein G conjugated tobeads. Subsequently, the individually precipitated VLPs were SDS-PAGEseparated and Western blot analyzed.

The VLPs precipitated with the 1918-HA Mabs were probed with a rabbitpolyclonal against the flu A/Viet/1023/04 (H5N1) and vice versa, theVLPs that were precipitated with the rabbit anti-HA5 (synthetic peptide)were probed with the 1918-HA Mabs. These experiments showed that the VLPprecipitated with the HA1918 Mabs did contain the HA5 molecules on theirsurfaces as demonstrated by positive Western blot when probed withfluA/Viet/1023/04 (H5N1) (FIG. 10).

Similarly, VLPs precipitated with anti-fluA/Viet/1023/04 (H5N1) andprobed on Western blots with the 1918-HA Mabs were also positive for the1918 HA demonstrating again that both HA molecules were present on thesurfaces of the same particle. The Western blot probed with thefluA/Viet/1023/05 (H5N1) rabbit polyclonal antibody showed, in additionto HA5, the other VLP structural components, flu proteins M1 and M2.(FIG. 10).

Electron Microscopy

To observe the morphology and confirm the surface antigenic compositionof the polyvalent VLPs, we examined purified polyvalent VLPs by negativestaining and dual immunogold-labeling electron microscopy. The negativestaining examination showed that the particles maintained an influenzavirus-like morphology with typical surface projections radiating fromthe entire surface. (FIG. 7A)

Furthermore, purified polyvalent VLPs were also examined by negativestaining after dual immuno-gold labeling. For these experiments,purified polyvalent VLPs were first incubated with a mixture ofanti-1819 HA (mouse Mabs) and anti-HA5 (rabbit anti-avian influenzaHA5-peptide) antibodies and subsequently incubated with a mixture ofanti-mouse (conjugate to 10 nm gold beads) and anti-rabbit (conjugatedto 5 nm gold beads). Immuno-gold labeled particles were then examined bynegative staining EM (FIG. 7B).

Hemagglutination/ELISA Assays

Studies were also performed to determine their activity inhemagglutination assays, which can be abrogated by the addition ofspecific antibody directed against either of the two HA moleculespresent on the surface of the particles.

In the first set of experiments, purified polyvalent influenza VLPvaccine was tested in an ELISA assay. In this study, purified polyvalentinfluenza virus-like particles were used as antigen to coat ELISAplates. Subsequently, different rows of the VLP coated plates wereincubated in triplicate with twofold serial dilutions of three differentspecific antibodies as follows: a) Anti-1918 HA mouse Mab (kindlyprovided by Dr. Peter Palese, Mount Sinai School of Medicine), b)Anti-HA5 rabbit polyclonal antibody (US Biological,), C) Anti-HA3 mouseMab (Roche Laboratory). Each group had a PBS row without primaryantibody as a control. After 3 washes the following horseradishperoxidase (HRP) conjugated secondary antibodies were added to thefollowing rows: The A and C rows (anti-1918 HA and anti-HA3 mouse Mabrespectively) received HRP conjugated goat anti-mouse and the row B(anti-HA3 rabbit polyclonal) HRP conjugated goat anti-rabbit, both fromBio-Rad Laboratories. The plates were then developed using one stepUltra TMB substrate (Pierce) and read on a plate reader at 450 nmabsorbance.

This study showed that the anti-1918 HA and anti-H5 antibodiesrecognized specific antigens on the polyvalent VLP vaccine which wasused as antigen to coat the ELISA plate (FIG. 8). On the other hand, theanti-HA3 antibody was unable to react with the polyvalent vaccine, asexpected, because the HA3 molecule was not included in the polyvalentVLP vaccine (FIG. 8). Thus, the two antigenically distinct HA moleculesare independently detected by both of two specific antibodies anti-1918HA and anti-HA5 whereas a non-specific anti-HA3 did not show binding onthe polyvalent VLP vaccine (FIG. 8).

In order to evaluate the ability of the polyvalent VLP vaccine toagglutinate red blood cells (RBC), a typical attribute of the influenzaviruses, we performed hemagglutination assays with purified polyvalentVLP vaccine and turkey red blood cells. This test showed that thepolyvalent VLPs were able to agglutinate red blood cells (first row, HAassay-no Ab in the hemagglutination inhibition test presented in FIG. 9)

Given the fact the polyvalent VLPs agglutinate RBC's, we postulated thatantibody against either of the two HA molecules incorporated on thesurface of the VLPs should have an inhibitory effect on thehemagglutination activity of the polyvalent VLP vaccine. To test thisassumption, we used the anti-1918 HA Mabs (described above) and a rabbitpolyclonal anti-HA5 Vietnam (kindly provided by Dr. Ruben Donis, CDC).Prior to the assay, each antibody was treated with the receptordestroying enzyme (RDE II Seiken, Accurate Chemicals, Westbury, N.Y.)and absorbed with turkey RBC to remove nonspecific agglutinins andinhibitors. Treated antibodies (1:5 final dilution after treatment) weretwo-fold serially diluted in a 96-well V-shaped bottom plate andsubsequently incubated with 4 HA units of polyvalent VLP. Followingpolyvalent VLP-antibody incubation, a 0.5% solution of turkey RBCs wasadded to the wells. Additional incubation was allowed and inhibition ofhemagglutination was observed when a sufficient amount of antibody,specific to the viral or VLP antigens, blocked VLP agglutination of theRBC, which is visualized by a RBC button at the base of the well (FIG.9).

This study showed not only that polyvalent VLPs are able to agglutinateturkey RBCs but also that a specific antibody against either of the twoHA molecules was able to partially inhibit the agglutinating activity ofthe particles. Furthermore, when the hemagglutination inhibition testwas carried out with a mixture of the two antibodies (anti-1918 HA plusanti-H5) the agglutination activity was completely abrogated (FIG. 9,complete inhibition). Non-specific antibodies, on the other hand, didnot abrogate the hemagglutination activity of the polyvalent VLPvaccine.

These experiments therefore, have clearly shown that the HA molecules onthe surface of the VLPs are functional in the agglutination of RBC whichcan be partially abolished by either one of the specific antibodies(anti-1918 HA or Anti-HA5) or completely eliminated by a mixture of thetwo (FIG. 9).

Thus, polyvalent influenza virus-like particles (VLPs) bearing on theirsurfaces two antigenically distinct HA molecules were prepared.

Example 2 Modification of Influenza M1 Protein-Encoding Sequences

A. Deletion and/or Mutation of Nuclear Localization Signal

The polynucleotide sequence encoding an M1 protein from a selectedinfluenza virus strain A/PR/8/34 is isolated. One or more of all thenuclear localization signal (positions 101-105, relative to wild-typeA/PR/8/34, having the amino acid sequence RKLKR (SEQ ID NO:1) aredeleted or modified.

B. Specific Mutations that Determine Spherical Sub-Viral StructureFormation

The morphology of wild type virus particles varies from an elongatedrod-shape to structures that are more or less spherical. Specificdeterminants on the sequence of the matrix proteins appear to influencethis morphological trait.

To maximize the assembly and release of homogeneous spherical sub-viralstructure vaccine, specific amino acids changes are introduced on theprimary sequence of the matrix proteins, for example, at amino acidpositions 41, 95, 98, 167, 204, 205 and/or 218 (FIG. 2).

Modification of Residues Involved in Monomers Contact and Sub-ViralStructure Yield

Viral matrix protein associates with the plasma membrane and establishescontact with other protein monomers forming an oligomerized scaffold,which together with the other components drives the release of thesub-viral structure from the cell of synthesis and assembly. Sitespecific changes as shown in Table 1 in the primary sequence of thematrix protein enhances protein-protein and protein membraneinteractions promotes higher specificity and better yield of thepolyvalent sub-viral structure vaccine.

Modification of One or More Sequence Specific Motifs (L-Domains)

Sequence specific motifs (L-domains) (FIG. 2) are critical in recruitinghost proteins which are necessary for the budding and pinching off ofthe sub-viral structure from the cell membrane. Insertion of one or moreL-domains at alternative locations within the sequence of the matrixproteins significantly enhances bud formation and release of sub-viralstructures from the surface of producing cells.

The DNA sequence coding for this modified matrix 1 (M1) protein is usedfor the production of each polyvalent vaccine utilizing this design.

Example 3 Modifications to Influenza Matrix Protein 2 (M2)

M2 protein is a trans-membrane protein that as a tetramer forms an ionchannel at the surface of the virus particle. The amount of M2 proteinincorporated into the wild type virus represents approximately 2% of theamount of M1 proteins.

Modifications to M2 are made as shown in FIG. 3, for example to theL-domains; and/or inclusion of sequences encoding immunomodulatorypolypeptides at the amino and/or carboxy terminals. Such modificationsincrease incorporation into the sub-viral structure vaccine and/ormodulate the type or potency of immune response elicited by thesub-viral structure vaccine by engineering hybrid M2 proteins that carryattached to its extra-cellular domain immune receptors,immune-modulators and/or adjuvant molecules.

Example 4 Sequences Encoding Chimeric HA and NA Polypeptides

As described above in Example 1, to aid in incorporation of the desiredantigens onto the surface of the sub-viral structure, the sequencesencoding the trans-membrane and cytoplasmic tail sequences of theselected HA- and/or NA-encoding sequences are replaced with thecorresponding HA and/or NA of influenza virus strain A/PR/8/34 virus.See, also, FIG. 4. This allows for better interaction with the plasmamembrane as well as with the underlying matrix proteins. In addition,identical trans-membrane and cytoplasmic domains in both surfacemolecules will aid in directing a similar level of incorporation ofcomponents into the sub-viral structure vaccine.

Example 5 Polyvalent Influenza VLPs

The sequences described above are introduced into one or more expressionvectors (e.g., baculovirus, plasmid) and introduced into suitable hostcells under conditions where polyvalent VLPs are formed. VLP formationis confirmed by Western blot analysis, neuraminidase assay and/orstandard hemagglutination assays. See, also, U.S. Patent Publication No.20050186621.

Example 5 In Vivo Vaccination with Polyvalent Influenza VLPs

The immunogenicity and protective efficacy of polyvalent VLP vaccinesare tested in BALB/C mice (Charles River Laboratories, Wilmington,Mass.). Mice are immunized intranasally or intramuscularly with singleor multiple doses of polyvalent VLPs as described herein.

What is claimed is:
 1. A noninfectious polyvalent influenza VLPcomprising an influenza M1 protein and three or more antigenic influenzaglycoproteins, wherein (i) the polyvalent VLP comprises two or moreantigenic hemagglutinin (HA) glycoproteins-derived from two or moredifferent Type A strains influenza strains and one or more antigenichemagglutinin (HA) glycoproteins-derived from a Type B strain; (ii) thepolyvalent VLP comprises less than all of the proteins of an influenzapolymerase complex; (iii) the three or more antigenic glycoproteins aredisplayed on the surface of the VLP.
 2. The VLP of claim 1, furthercomprising an M2 protein.
 3. The VLP of claim 2, wherein the M2 proteincomprises an amino acid modification as compared to a wild-type M2protein.
 4. The VLP of claim 1, further comprising an influenzanucleoprotein (NP).
 5. The VLP of claim 1, wherein the VLP furthercomprises one or more NA glycoproteins.
 6. The VLP of claim 1, whereinthe M1 protein comprises a nuclear localization signal and furtherwherein the nuclear localization signal (NLS) is modified by deletion,insertion or substitution of one or more residues.
 7. The VLP of claim1, wherein the M1 protein comprises one or more amino acids involved inprotein-protein interactions and further wherein one or more amino acidresidues involved in protein-protein interactions are modified.
 8. TheVLP of claim 1, wherein one or more L-domains are introduced into the M1protein.
 9. The VLP of claim 1, comprising a fusion of M1 and animmunomodulatory polypeptide.
 10. The VLP of claim 1, wherein at leastone of the antigenic glycoproteins is chimeric, wherein thetransmembrane domain of the at least one antigen glycoprotein isreplaced with a transmembrane domain from a different influenza stain.11. The VLP of claim 1, wherein at least one of the antigenicglycoproteins is chimeric, wherein the cytoplasmic tail region of theglycoprotein is replaced with a cytoplasmic tail region from a differentinfluenza stain.
 12. An isolated host cell comprising a polyvalent VLPaccording to claim
 1. 13. An isolated packaging cell for producing apolyvalent VLP according to claim 1, the packaging cell stably ortransiently transfected with one or more influenza protein-encodingsequences such that the polyvalent VLP is produced by the cell.
 14. Theisolated packaging cell line of claim 13, wherein a polynucleotideencoding M1 is stably transfected into the cell.
 15. The isolatedpackaging cell line of claim 13, wherein the cell is a mammalian cellline.
 16. An immunogenic composition comprising the polyvalent VLP ofclaim 1 and a pharmaceutically acceptable excipient.
 17. The immunogeniccomposition of claim 16, further comprising an adjuvant.
 18. A method ofproducing a VLP according to claim 1, the method comprising the stepsof: expressing one or more polynucleotides encoding the M1 and at leastthree influenza glycoproteins of the VLP in a suitable host cell underconditions such that the VLPs assemble in the host cell; isolating theassembled VLPs from the host cell.
 19. The method of claim 18, whereinthe host cell is selected from the group consisting of a mammalian cell,an insect cell, a yeast cell, a plant cell and a fungal cell.
 20. Themethod of claim 18, wherein the one or more polynucleotides areexpressed from an expression vector.
 21. The method of claim 20, whereinthe polynucleotides are operably linked to control elements compatiblewith expression in the selected host cell.
 22. The method of claim 20,wherein the expression vector is selected from the group consisting of aplasmid, a viral vector, a baculovirus vector and a non-viral vector.23. The method of claim 18, wherein one or more of the polynucleotidesare stably integrated into the host cell.
 24. A method of generating animmune response in a subject to three or more influenza viruses, themethod comprising the step of administering a composition comprising apolyvalent VLP according to claim 1 to the subject.
 25. The method ofclaim 24, wherein the composition is administered intranasally.
 26. Themethod of claim 24, wherein the composition is administered in amultiple dose schedule.